Our laboratory has shown that melatonin significantly inhibits the proliferation of ER+, but not ER- human breast cancer cells, and modulates the expression and transcriptional activity of the ERa. We have also demonstrated that melatonin can cross-talk with the retinoic acid (RA) signaling pathway, such that, when treated with a regimen of melatonin followed by RA at physiologic doses, breast cancer cells undergo apoptosis. In vivo, the combination of melatonin and 9-cis-RA was shown to be significantly more effective than RA alone at inhibiting the development and inducing the regression of carcinogen-induced rat mammary tumors. Furthermore, we have developed new data showing that melatonin's effects in tumor cells are mediated via the Mella/mt1 G protein-coupled receptor, and that overexpression of this receptor can enhance the response of ER+ breast tumor cells to the growth inhibitory effects of melatonin. These data led to our current hypothesis that the growth-inhibitory actions of melatonin are mediated, at least in part, through the Mella/mt1 melatonin receptor via modulation of the transcriptional activity of steroid/thyroid hormone receptor signaling pathways (ERa and RAR/RXR), and that overexpression of the Mella/mt1 receptor can generate a melatonin supersensitive phenotype in ER+ breast cancer cells. To test this hypothesis, we have developed the following Specific Aims: (1) To elucidate the importance of the Mella/mt1 receptor in the development and progression of breast cancer, and the signaling pathway(s) utilized by the Mel la/mt1 receptor to suppress MCF-7 cell proliferation; (2) To define the importance of the Mella/mt1 receptor in controlling breast cancer cell growth using Mel I a/mt1 gene ablation and transgenic overexpression techniques; (3) To determine if MCF-7 cells overexpressing the Mella/mtl receptor exhibit an enhanced response to the timed regimen of melatonin and RA, and to delineate further the interaction/cross-talk between melatonin and RA signaling pathways in regulating MCF-7 cell proliferation and apoptosis; and (4) To determine the optimal retinoid, dosage, and time period for the regimen of melatonin and RA which induces maximal regression of N-nitrosomethylurea (NMU)-induced rat mammary tumors. The characterization of the pathways by which melatonin inhibits the development and growth of breast tumors, and cross-talks with other hormone response pathways, such as the estrogen and retinoid pathways, is essential for the development of future endocrine strategies in the treatment and prevention of breast cancer.